Adeno xtm rapid titer kit

Large-scale virus preparation was performed using either CsCl gradients or by commercially available adenovirus purification kit (Adenopure, Puresyn, PA, USA). The titers of the viral stocks were determined using the Adeno-XTM Rapid Titer Kit (BD Biosciences, San Jose, CA, USA). Total DNA was harvested from 1 × 10⁶ infected cells using DNeasy blood and tissue kit (Qiagen, Germantown MD, USA) according to manufacturer’s protocol. 100 ng of DNA was used as a template for quantitative real-time PCR using primer sequences corresponding to adenoviral fiber and human β-Actin gene. For reverse transcription quantitative PCR, one microgram of total RNA was reverse transcribed using QuantiTect Reverse Transcription Kit (Qiagen). Sybr green–based real-time qRT-PCR was performed using SYBR Green RT qPCR SuperMix (Invitrogen) according to the manufacturer’s instructions. All reactions were done in triplicate. Standard curves were generated by serial dilution of each sample, and the relative amount of target gene mRNA was normalized to β-Actin mRNA using the following set of primers.